From shotgun to targeted proteomics: rapid Scout-MRM assay development for monitoring potential immunomarkers in Dreisse
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RESEARCH PAPER
From shotgun to targeted proteomics: rapid Scout-MRM assay development for monitoring potential immunomarkers in Dreissena polymorpha Maxime Leprêtre 1,2 & Mélissa Palos-Ladeiro 2 & Julien Faugere 1 & Christine Almunia 3 & Jérôme Lemoine 1 & Jean Armengaud 3 & Alain Geffard 2 & Arnaud Salvador 1 Received: 30 April 2020 / Revised: 27 July 2020 / Accepted: 6 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract A highly multiplexed liquid chromatography mass spectrometry–multiple reaction monitoring (MRM)-based assay has been developed for evaluating 107 candidate immune biomarkers in both hemocytes and plasma of the zebra mussel Dreissena polymorpha. The Scout-MRM strategy was employed for the first time, shortening the implementation of a targeted MRM bottom-up proteomics assay using selected immune protein-related peptides identified by shotgun discovery proteogenomics. This strategy relies on spiking scout peptides during the discovery phase and using them to build and deploy the MRM targeted proteomics method. It proved to be highly relevant, since about 90% of the targeted peptides and proteins were monitored and rapidly measured in both hemocyte and plasma samples. The sample preparation protocol was optimized by evaluating the digestion efficiency of tryptic peptides over time. The accuracy and precision of 50 stable isotope-labeled peptides were evaluated for use as internal standards. Finally, the specificity of the transitions was thoroughly assessed to ensure the reliable measurement of protein biomarkers. Several analytical and biological validation criteria were evaluated across hemocytes and plasma samples exposed ex vivo to biological contaminants, resulting in the validation of two Scout-MRM assays for the relative quantitation of 85 and 89 proteins in hemocytes and plasma, respectively.
Keywords Hemolymph . Bivalves . Immunomarkers . Multiplexed assay . Scout-MRM
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02868-2) contains supplementary material, which is available to authorized users. * Arnaud Salvador [email protected] 1
Université de Lyon, Université Claude Bernard Lyon 1, Institut des Sciences Analytiques, CNRS UMR 5280, 69100 Villeurbanne, France
2
Université de Reims Champagne-Ardenne, UMR-I 02 INERIS-URCA-ULH SEBIO Unité Stress Environnementaux et BIOsurveillance des milieux aquatiques, UFR Sciences Exactes et Naturelles, Campus du Moulin de la Housse, BP 1039, 51687 Reims, Cedex, France
3
Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI , 30200 Bagnols-sur-Cèze, France
Water quality evaluation in the framework of maintaining the sustainability of freshwater ecosystems and protecting biodiversity and ecosystem services represents a major challenge. These issues have led to the need to develop sensitive and predictive tools capable of assessing the toxicity of various anthropogenic contaminants in order
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